Among the many remarkable changes that occur in the developing red cell is the organization of a tissue specific, functionally unique cytoskeleton. This application focuses on the developmentally regulated genetic expression of one of these cytoskeleton proteins, Protein 4.1. Protein 4.1 is an 80 kd protein which cooperatively enhances the interaction of spectrin-actin in the cytoskeletal network and attaches it to the overlying lipid bilayer through a linkage with the transmembrane proteins band 3 (the anion channel) and glycophorins (sialoproteins carrying blood group specifications). Through these interactions, Protein 4.1 helps endow the red cell membrane with the viscoelastic interactions. Properties necessary to maintain its integrity in the face of circulatory stresses. Its clinical importance is exemplified by the hereditary hemolytic anemias thought to result from altered Protein 4.1 amount or structure. Multiple isoforms of Protein 4.1 are produced in a variety of tissues. My sponsor's laboratory has shown that these isoforms most likely arise from a single gene by alternative splicing of Protein 4.1 pre-mRNA. Moreover, terminally maturing red cells were found to produce a unique Protein 4.1 isoform which is expressed in a tissue specific and developmentally regulated manner. The goal of this training grant will be to investigate the factors mediating this tissue specific mRNA expression, thus enabling the applicant to gain expertise in RNA metabolism and chemistry, recombinant DNA methodology, protein chemistry, and cell biology. The applicant intends to carry out research under the direction of Dr. Edward Benz and Dr. Joan Steitz, focusing on the regulation of the tissue specific splicing of band 4.1 pre-mRNA in mouse erythroleukemia cells (MELC) (MEL cells have demonstrated the ability to express this erythroid specific isoform only after maturation is induced by specific agents.) My studies should provide me with broad training in cellular and molecular biology and, in addition, illuminate some of the molecular mechanisms underlying the role of alternative pre- mRNA splicing during normal and pathologic erythropoesis.